RESUMEN

Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

Asunto(s)

Neoplasias de la Mama/genética , Técnicas Electroquímicas/instrumentación , Reacción en Cadena de la Polimerasa Multiplex/instrumentación , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Técnicas Biosensibles/instrumentación , Mama/patología , Neoplasias de la Mama/diagnóstico , Femenino , Humanos , ARN Mensajero/análisis , Transcriptoma

RESUMEN

Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

Asunto(s)

Reacción en Cadena de la Polimerasa Multiplex/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Análisis de la Célula Individual/métodos , Estudios de Casos y Controles , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa Multiplex/normas , Células Neoplásicas Circulantes , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ARN

RESUMEN

The request of high specificity and selectivity sensors suitable for mass production is a constant demand in medical research. For applications in point-of-care diagnostics and therapy, there is a high demand for low cost and rapid sensing platforms. This paper describes the fabrication and functionalization of gold electrodes arrays for the detection of deoxyribonucleic acid (DNA) in printed circuit board (PCB) technology. The process can be implemented to produce efficiently a large number of biosensors. We report an electrolytic plating procedure to fabricate low-density gold microarrays on PCB suitable for electrochemical DNA detection in research fields such as cancer diagnostics or pharmacogenetics, where biosensors are usually targeted to detect a small number of genes. PCB technology allows producing high precision, fast and low cost microelectrodes. The surface of the microarray is functionalized with self-assembled monolayers of mercaptoundodecanoic acid or thiolated DNA. The PCB microarray is tested by cyclic voltammetry in presence of 5 mM of the redox probe K3Fe(CN6) in 0.1 M KCl. The voltammograms prove the correct immobilization of both the alkanethiol systems. The sensor is tested for detecting relevant markers for breast cancer. Results for 5 nM of the target TACSTD1 against the complementary TACSTD1 and non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A probes show a remarkable detection limit of 0.05 nM and a high specificity.

Asunto(s)

ADN/análisis , Técnicas Electroquímicas/instrumentación , Oro/química , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Biomarcadores de Tumor/análisis , Técnicas Biosensibles/instrumentación , Neoplasias de la Mama/diagnóstico , Sondas de ADN/química , Electrodos , Diseño de Equipo , Femenino , Humanos , Límite de Detección , Compuestos de Sulfhidrilo/química

RESUMEN

Long-term selection (> 45 generations) for low or high body weight from the same founder population has generated two extremely divergent lines of chickens, the low (LWS) and high weight (HWS) lines, which at the age of selection (56 days) differs by more than nine-fold in body weight. The HWS line chickens are compulsive feeders, whereas, in the LWS line, some individuals are anorexic and others have very low appetites. The involvement of the central nervous system in these behavioural differences has been experimentally supported. We compared a brain region at 0 and 56 days of age containing the major metabolic regulatory regions, including the hypothalamus and brainstem, using a global cDNA array expression analysis. The results obtained show that the long-term selection has produced minor but multiple expression differences. Genes that regulate neuronal plasticity, such as actin filament polymerisation and brain-derived neurotrophic factor, were identified as being differentially expressed. Genes involved in lipid metabolism were over-represented among differentially expressed genes. The expression data confirm that neural systems regulating feeding behaviours in these lines are different. The results suggest that the lines are set in separate developmental trajectories equipped with slightly different nervous systems. We suggest that the lines adapt behaviourally different to changing situations post hatch, such as the transition from dependence on yolk to feeding, in order to obtain energy. The present study has identified and exemplifies the kind of changes that may underlie the extreme differences in such behaviours.

Asunto(s)

Conducta Animal/fisiología , Peso Corporal/fisiología , Pollos/genética , Pollos/fisiología , Plasticidad Neuronal/genética , Factores de Edad , Animales , Peso Corporal/genética , Encéfalo/metabolismo , Cruzamiento , Conducta Alimentaria/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Masculino , Neurotransmisores/genética , Neurotransmisores/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Caracteres Sexuales

RESUMEN

BACKGROUND: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles. OBJECTIVE: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. METHODS: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. RESULTS: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. CONCLUSIONS: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

Asunto(s)

Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Antígenos HLA-DR/genética , Péptidos Cíclicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Artritis Reumatoide/genética , Autoantígenos/inmunología , Biomarcadores de Tumor/inmunología , Citrulina/inmunología , Colágeno Tipo II/inmunología , Reacciones Cruzadas , Proteínas de Unión al ADN/inmunología , Femenino , Fibrinógeno/inmunología , Genotipo , Cadenas HLA-DRB1 , Humanos , Inmunoglobulina A/biosíntesis , Masculino , Persona de Mediana Edad , Fosfopiruvato Hidratasa/inmunología , Proteínas Supresoras de Tumor/inmunología , Adulto Joven

RESUMEN

BACKGROUND: Basal cell carcinomas (BCCs) are prevalent tumours with uniform histology that develop without any known precursor lesion. Alterations in the sonic hedgehog-patched1 signalling pathway are accepted as necessary events for tumorigenesis, and mutations in the patched1 gene are frequently present in tumours. OBJECTIVES: To analyse transcript profiles in BCC. METHODS: We used laser-assisted microdissection to isolate and collect cell populations defined under the microscope. Peripheral cells from nests of BCC were selected to represent tumour cells, and normal keratinocytes from epidermis basal layer were used as control. Extracted RNA was amplified and hybridized on to a cDNA microarray. Results Our results show that BCC cells express a transcript signature that is significantly different from that of normal keratinocytes, and over 350 genes with various functions were identified as differentially expressed. The compiled data suggest an upregulation of the Wnt signalling pathway as a major event in BCC cells. Furthermore, tumour cells appear to have an increased sensitivity to oxygen radicals and dysregulated genes involved in antigen presentation. RESULTS: were validated at both the transcriptional level using real-time polymerase chain reaction and at the protein level using immunohistochemistry. CONCLUSIONS: We show that microdissection in combination with robust strategies for RNA extraction, amplification and cDNA microarray analysis allow for reliable transcript profiling and that antibody-based proteomics provides an advantageous strategy for the analysis of corresponding differentially expressed proteins. We found that expression patterns were significantly altered in BCC cells compared with basal keratinocytes and that the Wnt signalling pathway was upregulated in tumour cells.

Asunto(s)

Carcinoma Basocelular/metabolismo , Queratinocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Neoplasias Cutáneas/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Epidermis , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Microdisección/métodos , Persona de Mediana Edad , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología

RESUMEN

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.

Asunto(s)

Apoptosis , Hipoxia de la Célula , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Receptor fas/metabolismo , Secuencia de Bases , Sitios de Unión , Northern Blotting , Western Blotting , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/fisiología , Receptor fas/fisiología

RESUMEN

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

Asunto(s)

Chironomidae/genética , Biología Computacional , Células Epiteliales/metabolismo , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Proteínas de Insectos/genética , Transcripción Genética/genética , Animales , Línea Celular , Tracto Gastrointestinal/citología , Proteínas de Insectos/clasificación , Proteínas de Insectos/metabolismo , Mucosa Intestinal/citología

RESUMEN

We report the generation, assembly and annotation of expressed sequence tags (ESTs) from four chicken cDNA libraries, constructed from brain and testis tissue dissected from red junglefowl and White Leghorn. 21,285 5'-end ESTs were generated and assembled into 2,813 contigs and 9,737 singletons, giving 12,549 tentative unique transcripts. The transcripts were annotated using BLAST by matching to known chicken genes or to putative homologues in other species using the major gene/protein databases. The results for these similarity searches are available on www.sbc.su.se/~arve/chicken. 4,129 (32.9%) of the transcripts remained without a significant match to gene/protein databases, a proportion of unmatched transcripts similar to earlier non-mammalian EST studies. To estimate how many of these transcripts may represent novel genes, they were studied for the presence of coding sequence. It was shown that most of the unique chicken transcripts do not contain coding parts of genes, but it was estimated that at least 400 of the transcripts contain coding sequence, indicating that 3.2% of avian genes belong to previously unknown gene families. Further BLAST search against dbEST left 1,649 (13.1%) of the transcripts unmatched to any library. The number of completely unmatched transcripts containing coding sequence was estimated at 180, giving a measure of the number of putative novel chicken genes identified in this study. 84.3% of the identified transcripts were found only in testis tissue, which has been poorly studied in earlier chicken EST studies. Large differences in expression levels were found between the brain and testis libraries for a large number of transcripts, and among the 525 most frequently represented transcripts, there were at least 20 transcripts with significant difference in expression levels between red junglefowl and White Leghorn.

Asunto(s)

Encéfalo/fisiología , Pollos/genética , Etiquetas de Secuencia Expresada , Testículo/fisiología , Transcripción Genética , Animales , Pollos/clasificación , ADN Complementario/genética , Regulación de la Expresión Génica , Masculino , Sistemas de Lectura Abierta , ARN Mensajero/genética

RESUMEN

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Asunto(s)

Diferenciación Celular/fisiología , Neoplasias del Colon/patología , Neoplasias Ováricas/patología , Compuestos de Sulfhidrilo/metabolismo , Acetilcisteína/farmacología , Proteína Tirosina Quinasa CSK , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Neoplasias Ováricas/metabolismo , Fosfotransferasas/antagonistas & inhibidores , Fosfotransferasas/metabolismo , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Transactivadores , beta Catenina , Familia-src Quinasas

RESUMEN

BACKGROUND: The PTCH tumour suppressor gene is involved in the development of nearly all basal cell carcinomas (BCCs) of the skin and a fraction of squamous cell carcinomas (SCCs). A nonconservative Pro/Leu nucleotide polymorphism within PTCH exon 23 at codon 1315 was recently reported to be potentially important for the development of breast epithelial cell cancers. Objectives Accordingly, the status of PTCH codon 1315 was analysed for a possible association with the development of nonmelanoma skin cancers (NMSCs) in a pilot study. Because skin cancer risk is affected by specific population-dependent phenotypes such as skin and hair colour, codon 1315 was also analysed for normal allele frequency variation in human populations having differing extents of eumelanin vs. phaeomelanin. METHODS: The single nucleotide polymorphism in codon 1315 of the human PTCH gene was analysed in genomic DNA from six different populations comprising 472 blood samples and from 170 patients in four different categories with NMSC. Polymerase chain reaction and pyrosequencing were used to determine the allele frequencies. Allelic loss was furthermore determined in tumours following microdissection. RESULTS: The Pro/Pro genotype frequency ranged from 30% to 65% between populations, with a significant trend for a reduced frequency of the Pro/Pro genotype in populations having lighter pigmentation (P = 0.020). Pro/Pro frequency showed an increasing trend with increasing tumour case severity (P = 0.027). In 260 samples from 180 Swedish patients with NMSC and a control group of 96 healthy ethnically matched volunteers, no statistically significant pairwise differences between groups were detected in the PTCH codon 1315 allelic distribution, neither was a difference seen for multiple or early onset cases of BCC in the Swedish population. In Swedish patients with single tumours, allelic loss (loss of heterozygosity) was observed in 20 of 30 (67%) patients with BCC and four of 22 (18%) patients with SCC, with no preference in the allele lost. In contrast, the Pro/Pro genotype was frequent in seven U.S. patients having multiple independent BCCs. One of these patients was heterozygous, enabling allelic loss studies. Of 20 independent tumours, 11 had lost an allele; 10 of the 11 had lost Leu, suggesting nonrandom loss that favoured retention of Pro (P = 0.0059). CONCLUSIONS: Our results indicate an association between the eumelanin-to-phaeomelanin shift and a shift from the Pro/Pro genotype to Leu-containing genotypes. Failure to lose Pro during the shift to phaeomelanin may be associated with an increased population risk for BCC and increased individual risk for multiple BCC. During development of a tumour, the effect of Pro may be magnified by loss of the Leu allele.

Asunto(s)

Predisposición Genética a la Enfermedad , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/genética , Neoplasias Cutáneas/genética , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Codón/genética , Genotipo , Color del Cabello/genética , Humanos , Pérdida de Heterocigocidad , Receptores Patched , Receptor Patched-1 , Proyectos Piloto , Reacción en Cadena de la Polimerasa/métodos , Pigmentación de la Piel/genética

RESUMEN

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.

Asunto(s)

Pollos/genética , Etiquetas de Secuencia Expresada , Polimorfismo de Nucleótido Simple , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cartilla de ADN , Biblioteca de Genes , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Especificidad de la Especie , Testículo/metabolismo

RESUMEN

Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.

Asunto(s)

Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Línea Celular , Células Espumosas , Perfilación de la Expresión Génica/métodos , Humanos , Macrófagos , Reacción en Cadena de la Polimerasa , Receptores Citoplasmáticos y Nucleares/genética , Estadística como Asunto/métodos

RESUMEN

It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

Asunto(s)

Síndrome del Nevo Basocelular/genética , Proteínas de la Membrana/genética , Mutación/genética , Neoplasias Basocelulares/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Síndrome del Nevo Basocelular/patología , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Pérdida de Heterocigocidad/genética , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Neoplasias Basocelulares/patología , Receptores Patched , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Neoplásico/análisis , ARN Neoplásico/genética , Receptores de Superficie Celular

RESUMEN

The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

Asunto(s)

Árboles/crecimiento & desarrollo , Árboles/genética , Madera , Pared Celular/metabolismo , Celulosa/biosíntesis , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Lignina/biosíntesis , Meristema/genética , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética , Árboles/metabolismo

RESUMEN

Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light.

Asunto(s)

Genes p53/genética , Mutación , Fenómenos Fisiológicos de la Piel , Adulto , Anciano , Vestuario , Disección/métodos , Femenino , Humanos , Queratinocitos/fisiología , Terapia por Láser , Masculino , Protección Radiológica/métodos , Valores de Referencia , Piel/citología , Piel/efectos de la radiación , Luz Solar , Factores de Tiempo , Rayos Ultravioleta

RESUMEN

Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay. The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC). We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CIN) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.

Asunto(s)

Adenocarcinoma/virología , Carcinoma de Células Escamosas/virología , Genes p53/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Codón/genética , Femenino , Humanos , Infecciones por Papillomavirus/genética , Polimorfismo Genético , Análisis de Secuencia de ADN/métodos , Neoplasias del Cuello Uterino/genética , Displasia del Cuello del Útero/genética

RESUMEN

In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.

Asunto(s)

Cistina/análogos & derivados , ADN Complementario/genética , Perfilación de la Expresión Génica , Arteriosclerosis/genética , Arteriosclerosis/prevención & control , Línea Celular , Cistina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas Informáticos

RESUMEN

Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.

Asunto(s)

Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hormona de Crecimiento Humana/farmacología , Hipófisis/fisiología , Animales , Corazón/fisiología , Humanos , Hipofisectomía , Riñón/fisiología , Hígado/fisiología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.

Asunto(s)

Proteínas de Fusión Oncogénica/genética , Proteínas/genética , Empalme del ARN , Sarcoma Sinovial/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Línea Celular Transformada , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonación Molecular , Regulación hacia Abajo , Exones , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Represoras , Alineación de Secuencia , Testículo/fisiología , Transcripción Genética